Medrogestone production

ABSTRACT

A process for the production of 6,17α-dimethylpregna-4,6-diene-3,20-dione which comprises isolating 3β,5α, 6β-trihydroxy- 6α,17α- dimethylpregnan-20-one monomethanolate from the solution of a Grignard reaction product of 3β,5α-dihydroxy -17α-methyl-17β-carbomethoxyandrostane-6-one and a methylmagnesium halide by solvent displacement with methanol; oxidizing the monomethanolate to the 3,20-dione; dehydrating the said 3,20-dione to the 4-ene-3,20-dione; dehydrating the 4-ene-3,20 dione; removing a 6-methylene-4-ene-3,20-dione byproduct and isolating the desired product.

RELATED APPLICATION

This is a division of application Ser. No. 08/239,187, filed May 5,1994, now U.S. Pat. No. 5,428,181, by Michael W. Winkley and Robert D.Mitchell.

BACKGROUND OF THE INVENTION

Medrogestone (6,17α-dimethylpregna-4,6-diene-3,20-dione) is a knownprogestogen useful in hormone replacement therapy for inducing andreestablishing normal menstrual cycles (irregular cycles, secondaryamenorrhea, oligoamenorrhea, etc.) , assuring regular endometrialshedding and in arresting and controlling dysfunctional uterine bleeding(menhorrhagia, metrorrhagia, etc.). Medrogestone has been used alone andin conjunction with sequential estrogen treatment.

Medrogestone (6,17α-dimethylpregna-4,6-diene-3,20-dione) isconventionally prepared by the Grignard reaction of methyl magnesiumbromide with3β-acetoxy-5α-hydroxy-17αor-methyl-17β-carbomethoxyandrostan-6-one toobtain 3β,5α,6β-trihydroxy -6α,17α-dimethyl-17β-carbomethoxyandrostane.Upon further reaction with methyl magnesium bromide, the correspondingpregnan-20-one is obtained. The, thusly, produced pregnan-20-one isoxidized with 8N chromic acid to yield50α,6β-dihydroxy-6α,17α-dimethylpregnan-3,20-dione which is convertedinto medrogestone by acid catalyzed dehydration (U.S. Pat. No. 3,170,936Examples 3-6). This process leads to the production of three impuritieswhich amount to about five percent of the product. These impurities canbe removed from medrogestone with some difficulty and at considerableadditional expense. Hence, avoidance of the production of thesebyproducts during the production of medrogestone would provide amarkedly improved method for the manufacture of this valuable medicinalcompound.

It is currently believed that the three major contaminants formed duringthe preparation of medrogestone are: (1)6,17α-dimethyl-17β-isopropenylandrosta-4,6-dien-3-one (generated bymethylation of the 17β-methoxycarbonyl group to give the17β-1-hydroxy-1-methylethyl substituent which is dehydrated along withthe 5α- and 6β-hydroxyl groups by the conventional acid catalyzedprocess used in the final step of production); (2)6-methylene-17α-methylpregn-4-ene-3,20-dione and (3)6,17α-dimethylpregna-6,8(14)-diene-3,20-dione, each of which are formedas rearrangement products of the acid catalyzed dehydration of the5α,6β-dihydroxy intermediate.

DESCRIPTION OF THE INVENTION

In accordance with this invention, there is provided a process for theproduction of 6,17α-dimethylpregna-4,6-diene-3,20-dione which comprises(a) isolating 3β,5α,6β-trihydroxy-6α,17α-dimethylpregnan-20-onemonomethanolate from the solution of a Grignard reaction product of3β,5α- dihydroxy-17α-methyl-17β-carbomethoxyandrostane-6-one and amethyl magnesium halide by solvent displacement with methanol;

(b) oxidizing 3β,5α,6β-trihydroxy-6α,17α-dimethylpregnan-20-onemonomethanolate to obtain5α,6β-dihydroxy-6α,17α-dimethylpregnan-3,20-dione;

(c) dehydrating 5α,6β-dihydroxy-6α,17α-dimethylpregnan-3,20-dione underalkaline conditions to obtain6β-hydroxy-6α,17α-dimethylpregn-4-ene-3,20-dione;

(d) dehydrating 6β- hydroxy-6α,17α- dimethylpregn-4-ene-3,20-dione withboron trifluoride etherate at reaction and work-up temperatures belowabout 65° C.;

and (e) isolating 6,17α-dimethylpregna-4,6-diene-3,20-dione from thediene by-product, 6-methylene-17α-methylpregn-4-ene-3,20-dione, presentin the reaction product mixture from step (d) by reaction with maleicanhydride to obtain: ##STR1## hydrolyzing the Diels-Alder adduct in thepresence of a base to form the dicarboxylic acid salt: ##STR2## andseparating of the salt by aqueous phase separation from a non-aqueoussolvent containing 6,17α-dimethylpregna-4,6-diene-3,20-dione.

It has now been discovered that, by forcing 3β,5α,6β-trihydroxy-6α,17α-dimethylpregnan-20-one from the reaction solutionresulting from the methylation of 3β,5α-dihydroxy-17α-methyl-17β-carbomethoxyandrostane-6-one under Grignard conditions anddrying the crystalline product thus formed, a stable monomethanolsolvate is obtained free from methylated impurities. In addition,carefully controlling the dehydration of5α,6β-dihydroxy-6α,17α-dimethylpregnan -3,20-dione under alkalineconditions in the presence of methanol, provides the novel intermediate6β- hydroxy -6α,17α-dimethylpregn-4-ene-3,20-dione in good yields,which, unlike 5α,6β-dihydroxy-6α,17α-dimethylpregnan-3,20-dione, isreadily crystallized from lower alkanols such as methanol, ethanol,isopropanol, etc., to provide pure intermediate product. Even withoutfurther purification, very little impurity appears with this newintermediate, and it is sufficiently pure to directly undergo furtherdehydration to Medrogestone. If the conversion of 6β- hydroxy-6α,17α-dimethylpregn-4-ene-3,20-dione into Medrogestone is carded outwith the Lewis acid, boron trifluoride etherate, rather than with themore conventional agents such as SOCl₂ /pyridine, POCl₃ /pyridine orMsCl/Et₃ N/DMAP/CH₂ Cl₂, reaction temperatures below about 65° C. arefeasible and isomerization to6,17α-dimethylpregna-6,8(14)-diene-3,20-dione is largely avoided.Medrogestone, prepared in accordance with this invention, does containan identifiable amount of 6-methylene -17α-methylpregn-4-ene-3,20-dionewhich is readily removed by reaction of the impurity (diene) with maleicanhydride (dienophile) to produce the Diels-Alder adduct which can thenbe hydrolyzed in the presence of a base to afford the dibasic acid saltthat is easily removed from the desired product by aqueous extraction.Medrogestone produced in accordance with the process of this inventionexhibits an HPLC strength in the order of 99.2% with total impurities atabout 0.08% , thereby providing a very pure therapeutic agent.

The novel process of this invention is illustrated by the followingexamples:

EXAMPLE 1 3β,5α,6β-Trihydroxy-6α,17α-dimethylpregnan-2-one

To a 3M solution of methyl magnesium chloride in tetrahydrofuran (1.777L, 5.33 moles) plus 50 mL tetrahydrofuran used as a transfer rinse,under a nitrogen atmosphere, cooled to 0°-5° C. was added a slurry of3β,5α-dihydroxy-17α-methyl -17β-carbomethoxyandrostane-6-one (200 g.,0.528 mole) in tetrahydrofuran (1.52 L)over a period of 30 minutes,while maintaining the temperature below 25° C. Addition of the slurrywas completed with a rinse of tetrahydrofuran (120 mL). The temperatureof the vigorously stirred reaction mixture was brought to 60° C. Afterapproximately one hour the dark solution thickened and stirring becamedifficult. After approximately two hours the reaction mixture became astirrable gray slurry and it was kept at 60° C. for a total of nineteenhours. The mixture was cooled to 0°-5° C. and a solution of ammoniumchloride (176.0 g) in water (700 mL) was cautiously added over a periodof one hour, while maintaining the temperature below 30° C. To theresulting slurry was added water (880 mL) and the mixture was stirredfor ten minutes. To the cooled mixture was added 500 mL of 12Nhydrochloric acid over a period of fifteen minutes, while maintainingthe temperature between 15° and 20° C. The mixture was then stirred atroom temperature (23°-27° C.) for one hour. The two resulting clearphases were separated and the organic phase was washed with 80%saturated brine (2×480 mL). The organic phase was washed a third timewith 80% saturated brine (480 mL) and the pH of this third wash wasadjusted to pH 7 during the wash with 5% sodium bicarbonate solution.The resulting mixture of organic and aqueous phases was filtered toremove any remaining solid material and the phases were separated again.The organic phase (3,200 mL) was distilled to 1200 mL. The distillationwas continued while adding methanol (2.400 L) dropwise to maintain thevolume at 1200 mL while azeotropic ally removing most of thetetrahydrofuran solvent. The resulting slurry of crystals in methanolwas allowed to cool to 30° C. over a period of 30 minutes and thencooled to 0°-5° C. over a period of 30 minutes. After forty five minutesat 0°-5 ° C. the crystals were collected on a filter and washed with0°-5° C. methanol (2×200 mL). Drying under oil pump vacuum at 45°-50° C.for two hours gave 3β,5α,6β-trihydroxy -6α,17α-dimethylpregnan-20-one asa 99% pure, stable monomethanolate, m.p.=235.0°-240.5° C. High PressureLiquid Chromatography established the purity of this product as:Strength=101.6% and Impurity=0.82%.

EXAMPLE 2 5,60 ,6β-Dihydroxy-6α,17α-dimethylpregnan-3,20-dione

To a stirred suspension of the compound produced in Example 1 (75.0 g,0.183 mole) in acetone (750 mL) cooled to 0°-5° C. in an ice/acetonebath, was added 158 mL (0.422 moles) of a 2.67M solution of Jones'reagent [Reagents for Organic Synthesis, Wiley Interscience, vol. 1,page 142, 1967), while maintaining the temperature below 10° C., over aperiod of one hour. The addition was completed with a rinse (8 mL) ofwater. The resulting mixture was stirred at 0°-5° C. until the reactionwas complete (about two hours) as judged by thin layer chromatography onsilica gel GF₂₅₄ (E. Merck) plates with chloroform-methanol (100:8) asdeveloper (visualization by sulfuric acid charring). The stirred greenmixture was neutralized to pH 2.5 by dropwise addition of approximately210 mL of 6% w/w sodium hydroxide while maintaining the temperaturebelow 5° C. The mixture was stirred at ambient temperature for half anhour. Water (565 mL) was added and the mixture was extracted withdichloromethane (450 mL and 225 mL). The combined organic extracts werewashed thrice with acidified water [first, with 565 mL H₂ O to which 30mL 12N HCl was added; second, with 300 mL H₂ O to which 15 mL HCl wasadded and third, with 300 mL H₂ O to which 15 mL HCl was added]. Theorganic phase was washed with water (300 mL) and the pH of the aqueousphase was adjusted during the wash to a value of 7 with 10% NaHCO₃solution. The dichloromethane solution was washed one more time withwater (300 mL) and the pH of the wash checked for neutrality. Thesolution was distilled to a volume of 300 mL so that a thick slurry ofcrystals was obtained. Toluene (150 mL) was added to bring the volume to450 mL. The mixture was distilled and the volume maintained at 450 mL bythe dropwise addition of toluene (300 mL). When all the toluene had beenadded the volume was reduced to 300 mL and the stirred slurry wasallowed to cool to room temperature over a period of two hours. Afterstirring the slurry at room temperature for 2 hours, the crystals werecollected on a filter and washed with 0°-5° C. toluene (3×55 mL). Dryingat 65° C. for 18 hours gave 63.70 g. (92.7% of theory) of 5α,6β-dihydroxy-6α,17α-dimethylpregnan-3,20-dione as a pale yellow product,having a melting point of 234.8°-237.2° C. dec. This product, 95.5%:pure by HPLC, was considered sufficiently pure for conversion into6β-hydroxy-6α,17α-dimethylpregn-4-ene-3,20-dione.

EXAMPLE 3 6β-Hydroxy-6α,17α-dimethylpregn-4-ene-3,20-dione

A mixture of the compound produced in Example 2 (620.00 g), methanol(8.00 L) and 500 mL of 5N NaOH was mechanically stirred and heated atreflux (69°-70° C. under a nitrogen atmosphere for one hour. Theresulting solution was allowed to cool to 65° C. and maintained at thattemperature while adding water (3,250 mL) over a period of 35 minutes.The resulting slurry of crystals was cooled to 20° C. over a period ofone hour. The white crystals were collected on a filter and washedconsecutively with 3:7 methanol-water (3×1 L) and water (2×1 L). Dryingat 80°-90°C. in a vacuum for 42 hours gave 514.98 g. (87.2% of theory)of crude product. Recrystallization of the crude product (314.62 g) from2- propanol afforded 292.83 g. (93.1% of theory) of pure title compound;m.p=235°-237.8° C., [α]_(D) =26.4° (c=1, CHCl₃); M+(DEI) 358, λmax.(KBr) 1668, 1691 cm⁻¹, pmr (DMSO-d₆) δ5.78 (vinylic proton at C-4).

Elemental analysis for C₂₃ H₃₄ O₃ Calc'd: C, 77.05; H, 9.56 Found: C,76.84; H, 9.49

EXAMPLE 4 6,17α-Dimethylpregna-4,6-diene-3,20-dione (Medrogestone)

A solution of the dried, unrecrystallized 6β-hydroxy-6α,17α-dimethyl-pregn-4-ene-3,20-dione as prepared in Example 3 (72.00 g., 0.2008 mole)in methylene chloride (650 mL) was distilled until 50 mL of distillatewas collected. To the stirred, cooled (22° C.) solution under a nitrogenatmosphere was added dropwise, boron trifluoride etherate (50 mL, 0.4065moles, 2.02 molar excess). The mixture was warmed to reflux over aperiod of one hour (a white crystalline complex precipitated during thistime). The mixture was maintained at reflux until the reaction wascomplete (about one hour) as judged by thin layer chromatography onsilica gel (E. Merck) plates using methylene chloride: ethyl acetate(1:1) as developer. The resulting stirred, dark red solution was cooledto 10°-15° C. using an ice bath and a slurry of NaHCO₃ (100 g) in H₂ O(450 mL) was cautiously added over a period of five minutes. The twophase mixture was stirred vigorously at 10° -15° C. for one hour, at15°-20° C. for one hour and at 20°-25° C. for thirty minutes. The layerswere separated and the organic phase was washed thrice with water (250mL). The methylene chloride solution was distilled to 150 mL (final pottemperature 50° C.) and toluene (100 mL) was added. The solution wasdistilled under a water aspirator vacuum to 150 mL once more. Toluene(450 mL) was added and the solution was distilled under water aspiratorvacuum to a volume of 480 mL. This low temperature synthesis and work upavoids the production of 6,17α-dimethylpregna-6,8(14)-diene-3,20-dionewhich is believed to be a thermodynamically produced byproduct.

To the resulting toluene solution of crude product was added maleicanhydride (11.0 g, 0.1122 mole) and the mixture was heated to reflux(pot temperature 114°115° C.) under nitrogen for one hour underDiels-Alder conditions. The cooled (22° C.) solution was added to astirred solution of sodium carbonate (40 g) in water (720 mL) also at22° C. and the stirred mixture heated to 50° C. and maintained at 50° C.for one hour. The mixture was cooled to room temperature (25° C.) andthe layers were separated. The organic phase was washed consecutivelywith 200 mL (×2) and 100 mL (×1) of 1.5N sodium hydroxide, 300 mL ofsaturated brine and 200 mL (×2) of water. At this stage the aqueouswashings were neutral. The solution was distilled under a wateraspirator vacuum to near dryness. Heptane (250 mL) was added and thestirred mixture was heated to reflux (98°-99° C.) under nitrogen. Theresulting solution was allowed to cool and the title compoundcrystallized by appropriate seeding. Crystallization started at 78° C.When the temperature of the stirred slurry of crystals reached 35° C.ice/water cooling was used to lower the temperature to 0°-5° C. Thecrystals were collected on a filter and washed with heptane (2×75 mL);55.2 g, (80.8% of theory).

Three crystallizations from 2-propanol afforded pure material (38.03 g,55.6% ) having a m.p.=145°-147° C., [α]_(D) =77.7° (c=1, CHCl₃), M⁺(PBEI) 340 and λmax. (KBr) 1665, 1691 cm⁻¹.

Elemental analysis for C₂₃ H₃₂ O₂ Calc'd: C, 80.94; H, 9.26 Found: C,80.90; H, 9.28

In addition to the novel process, it has been found that the novelintermediate, 6β-hydroxy-6α,17α-dimethylpregn-4-ene-3,20-dione is usefulin preventing smooth muscle cell proliferation. This utility wasestablished by subjecting the compound to the following standardexperimental test procedure:

Primary rat aorta smooth muscle cell cultures grown in media 199 (Gibco)plus 10% fetal bovine serum (Gibco) were washed with calcium, magnesiumfree Dulbecco's phosphate buffered saline and trypsinized for fiveminutes. Cells were scraped from culture dishes with a rubber policemanand centrifuged. Cells were resuspended in M199 plus 10% fetal bovineserum containing [³ H)-thymidine (0.5 μCi/mL) at 8-15,000 cells/mL, andwere placed into plate wells. The novel intermediate was added to eachwell and the plates were incubated for 24 hours at 37° C. in a 5% CO₂atmosphere. The cultured cells were treated with trichloracetic acid toremove acid soluble proteins, leaving only cell superstructure and DNA.These cells were solubilized, counted by scintillation and the resultscompared with control to determine the number of cells per well, thedifference in cells being expressed as a percent of control. Based uponthis study, 6β-hydroxy-6α,17α-dimethylpregn -4-ene-3,20-dionedemonstrated an IC₅₀ of 26.45 μM whereas Medrogestone demonstrated anIC₅₀ of 14.84 gM.

Hence, the compound, 6β-hydroxy-6α,17α-dimethylpregn-4-ene -3,20- dione,as an inhibitor of smooth muscle proliferation, is useful in thetreatment of restinosis and atherosclerosis and it and pharmaceuticalcompositions containing it, present additional aspects of thisinvention. The pharmaceutical compositions involved may be administeredby any route conventionally employed with steroids, for example,transdermally, orally, parenterally, intranasally, etc. The steroid maybe administered neat or in combination with a pharmaceuticallyacceptable carrier.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,glidants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carrier is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredient. Suitablesolid carriers include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine,low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carrier can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The compound can also be administered orallyeither in liquid or solid composition form.

The dosage requirements will vary with the route of administration, theseverity of the symptoms presented and the particular subject beingtreated. Based on the results obtained in the standard pharmacologicaltest procedure, projected daily dosages of6β-hydroxy-6α,17α-dimethylpregn-4ene-3,20-dione, would be 0.005-50 mg/kgand preferably between 0.05-10 mg/kg.

Treatment will generally be initiated with small dosages less than theoptimum dose of the compound. Thereafter the dosage is increased untilthe optimum effect under the circumstances is reached; precise dosagesfor oral, parenteral, intranasal, or transdermal administration will bedetermined by the administering physician based on experience with theindividual subject treated.

What is claimed is:
 1. A process for the production of6,17α-dimethylpregna-4,6-diene-3,20-dione which comprises (a) isolating3β,5α,6β-trihydroxy-6α,17α-dimethylpregnan-20-one monomethanolate fromthe solution of a Grignard reaction product of3β,5α-dihydroxy-17α-methyl-17β-carbomethoxyandrostane-6-one and amethylmagnesium halide by solvent displacement with methanol;(b)oxidizing 3β,5α,6β-trihydroxy-6α,17α-dimethylpregnan-20-onemonomethanolate to obtain 5α,6β-dihydroxy-6α,17α-dimethylpregnan-3,20-dione; (c) dehydrating5α,6β-dihydroxy-6α,17α-dimethylpregnan-3,20-dione under alkalineconditions to obtain 6β-hydroxy-6α,17α-dimethylpregn-4-ene -3,20-dione;(d) dehydrating 6β-hydroxy-6α,17α-dimethylpregn-4-ene-3,20 -dione withboron trifluoride etherate at reaction and work-up temperatures belowabout 65° C.; (e) isolating 6,17α-dimethylpregna-4,6-diene-3,20-dionefrom the diene by-product, 6-methylene-17α-methylpregn-4-ene-3,20-dione,present in the reaction product mixture of step (d) by reaction withmaleic anhydride, (f) hydrolyzing the Diels-Alder adduct in the presenceof a base to form the dicarboxylic acid salt and (g) separating the saltby aqueous phase separation from a non-aqueous solvent containing6,17α-dimethylpregna-4,6-diene-3,20-dione.
 2. The process of claim 1 inwhich the base employed in the hydrolysis of the Diels-Alder adduct isan alkali metal hydroxide.